Method for detecting heat-resistant micro-organisms capable of contaminating certain food products

ABSTRACT

A method for detecting Byssochlamys nivea, Neosartorya fischeri and Zygossaccharomyces bailii micro-organisms by amplifying the genomic DNA of the targeted micro-organisms using as a primer a sequence contained on the internal transcript spacers (ITS) of the ribosomal unit, namely, for Byssochlamys nivea, on ITS1 corresponding to SEQ ID 1 and on ITS2 corresponding to SEQ ID 2; for Neosartorya fischeri, on ITS1 corresponding to SEQ ID 3, and on ITS2 corresponding to SEQ ID 4; and for Zygosaccharomyes bailii, on ITS1 corresponding to SEQ ID 5 and on ITS2 corresponding to SEQ ID 6.

The instant application is a National Phase Application of International Application PCT/FR96/00821, filed May 31, 1996.

FIELD OF THE INVENTION

The present invention relates to a method for the rapid detection of heat-resistant eukaryotic microorganisms capable of contaminating certain products in the food sector, particularly fruit-based products.

BACKGROUND OF THE INVENTION

More particularly, the microorganisms studied are two filamentous fungi, Byssochlamys nivea and Neosartorya fischeri and a yeast, Zygosaccharomyces bailii.

These contaminants of food products can develop on the fruit-based products, giving them, particularly for Byssochlamys nivea, unpleasant notes of the plastic or antiseptic type; the growth of Neosartorya fischeri is responsible for spoilage and the appearance of mycotoxins, whereas as that of Zygosaccharomyces bailii causes a formation of gas.

These microorganisms particularly in the form of spores, exhibit a very high heat resistance which allows them to persist, even after conventional pasteurization treatments, which are applied in the food industry.

The existing tests allowing the detection of these microorganisms consist in altering the samples of a selective medium by taking advantage of, as a selection factor, the heat resistance of the filamentous fungi or the resistance of the yeast to certain preservatives conventionally used in the sector, such as benzoic acid. However, this type of detection involves cultures and, what is more, fairly long cultures, namely from several days to several weeks, which makes them practically unusable in the food sector since they could involve preserving the products, which may be contaminated, for a very long time.

The search is therefore underway for a detection test allowing the very rapid detection of these microorganisms, it being possible for the said test to be carried out in a few hours at the most, in order to find out rapidly if the sampled product can continue or otherwise to be treated in the subsequent stages.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention proposes a process allowing the detection of Byssochlamys nivea, Neosartorya fischeri and Zygosaccharomyces bailii by amplification of the genomic DNA of the targeted microorganisms using, as primers, a pair of sequences contained in the internal transcribed spacers (ITS) of the ribosomal unit:

for Byssochlamys nivea,

on ITS1 corresponding to SEQ ID NO: 1,

on ITS2 corresponding to SEQ ID NO: 2,

for Neosartorya fischeri,

on ITS1 corresponding to SEQ ID NO: 3,

on ITS2 corresponding to SEQ ID NO: 4,

for Zygosaccharomyces bailii

on ITS1 corresponding to SEQ ID NO: 5,

on ITS2 corresponding to SEQ ID NO: 6.

In the ITS1 and ITS2 sequences, there will be chosen more particularly,

for Byssochlamys nivea, the sequence SEQ ID NO: 7 and SEQ ID NO: 8,

for Neosartorya fischeri SEQ ID NO: 9 and SEQ ID NO: 10,

for Zygosaccharomyces bailii SEQ ID NO: 11 and SEQ ID NO: 12.

Among the amplification methods which can be used, the so-called PCR (Polymerase Chain Reaction) method will be used more particularly.

The tests, reported in particular in the examples, show that these primers are perfectly discriminating and make it possible to distinguish between the desired microorganisms and the microorganisms, which are even very close, and related.

The present invention also relates to a process for detecting these microorganisms in food products, particularly with fruit-based food products and more particularly strawberry-based food products, in which the sample containing the fruits is pretreated so as to liberate and concentrate the spores or the cells, to reduce or suppress the action of the inhibitors of Taq Polymerase and to extract the DNA from the spores or cells.

More particularly, the samples containing solid products will be treated with a mixture of cellulase/hemicellulase so as to liquefy them and then filtered and centrifuged under conditions which make it possible to obtain a product containing the cells from which the DNA can be extracted.

Since the samples contain most often inhibitors of Taq Polymerase, it is necessary to provide for a stage which makes it possible to reduce the activity of the inhibitors, either by dilution, or by treatment of the sample with phenol-chloroform and precipitation with alcohol.

Although the present invention is more particularly intended for the food industry, it is evident that it can also be used to detect the microorganisms involved in other samples which might not have any relationship with the food industry.

Finally, the present invention relates to primers, as defined above, as well as detection kits using the said primers.

Other characteristics and advantages of the present invention will emerge on reading the examples below.

EXAMPLE 1 Procedure for Extraction of DNA and for Amplification

I) Starting with about 5 mm² of fungal mycelium, 10⁵ fungal spores or 10⁶ yeast cells:

in a screw-top microtube (1.5 ml Eppendorf type), suspension of the microorganisms in 200 μl of 50 mM Tris buffer containing 20 mM EDTA, 0.8% SDS, pH 8.5, to which there are added 100 μl of glass beads (diameter 0.25-0.5 mm),

vortex for 1 minute at maximum frequency,

incubation of the mixture for 15 minutes at 100° C. (boiling water),

for the treatment of the spores, the vortexing and the incubation are replaced with:

freezing in liquid N², boiling 100° C. 5 minutes,

refreezing in liquid N², boiling 100° C. 10 minutes,

centrifugation 1 minute at 10,000 g (bench centrifuge),

recovery of the supernatant

10-fold dilution of the supernatant in water.

This diluted supernatant is used directly for the amplification.

(II) The amplification is carried out in a final volume of 25 μl containing 20 mM Tris-HCl pH 8.5, 16 mM (NH₄)₂ SO₄, 2.5 mM MgCl₂, 150 μg/ml of bovine serum albumin, 0.2 μM of each dNTP and 100 μM of each of the primer oligonucleotides. Two units of Taq DNA polymerase (BioTaq, Bioprobe systems, France) are used by reaction. The amplification reaction is carried out in the Perkin Elmer 2400 thermocycler (Perkin Elmer Corp., U.S.A.) in the following manner:

for Zygosaccharomyces, Byssochlamys and Neosartoria

15 seconds at 94° C.

10 seconds at 58° C.

20 seconds at 72° C.

This cycle is repeated 30 times. It is followed by a 5 minute terminal extension at 72° C.

(III) The products obtained are visualized under UV after migration on an electrophoretic gel having the following composition: 1×TBE buffer, 1 μg/ml ethidium bromide, 0.8% agarose.

EXAMPLE 2

The results observed using the process according to the present invention on various strains close to the strains to be detected showing the excellent discrimination provided by the claimed sets of primers will be found below.

The primers used are:

SEQ ID 7 and 8 for Byssochlamys nivea,

SEQ ID 9 and 10 for Neosartorya fischeri,

SEQ ID 11 and 12 for Zygosaccharomyces bailii.

It should be noted that A. fumigatus responds to the probe because it is the asexual form of N. fischeri.

    ______________________________________                                         Primer oligonucleotides specific for Neosartoria fischeri                                                       T ° C.                                                                        PCR                                       Genus Species Strain hy. response                                            ______________________________________                                         Aspergillus                                                                             floriformis                                                                               M 93 2663  57° C.                                                                        negative                                    Aspergillus fumigatus M 70 665 57° C. positive                          Aspergillus fumigatus M 88 2521 57° C. positive                         Byssochlamys nivea M 90 1496 57° C. negative                            Byssochlamys nivea M 93 2932 57° C. negative                            Emericella nidulans M 88 2519 57° C. negative                           Emericella nidulans M 84 2558 57° C. negative                            var. acristata                                                                Emericella nidulans M 84 2556 57° C. negative                            var. dentata                                                                  Emericella nidulans M 68 1987 57° C. negative                            var. lata                                                                     Emericella nidulans M 84 9557 57° C. negative                            var. shinulata                                                                Eupenicillium brefeldianum M 89 2573 57° C. negative                    Eurotium amstelodami M 50 142 57° C. negative                           Eurotium amstelodami M 88 2536 57° C. negative                          Eurotium repens M 66 2534 57° C. negative                               Mariannae elegans M 95 3777 57° C. negative                             Monascus ruber M 65 1079 57° C. negative                                Neosartoria fennelliae M 93 2982 57° C. positive                        Neosartoria fennelliae M 95 3781 57° C. positive                        Neosartoria fennelliae M 953780 57° C. positive                         Neosartoria pseudofischeri M 93 2986 57° C. positive                    Neosartoria pseudofischeri M 95 3784 57° C. positive                    Neosartorya aurata M 93 2980 57° C. positive                            Neosartorya aureola M 90 3623 57° C. positive                           Neosartorya aureola M 93 2981 57° C. positive                           Neosartorya aureola M 95 3785 57° C. positive                           Neosartorya fennelliae M 93 2983 57° C. positive                        Neosartorya fischeri M 90 2660 57° C. positive                          Neosartorya fischeri M 88 2482 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 89 2707 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 90 3618 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 90 3619 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 90 3620 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 90 3621 57° C. positive                           var. fischeri                                                                 Neosartorya fischeri M 87 3513 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 88 2480 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 88 3577 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 90 3622 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 92 2891 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 92 2925 57° C. positive                           var. glabra                                                                   Neosartorya fischeri M 88 2480 57° C. positive                           var - spinosa                                                                 Neosartorya fischeri M 88 3574 57° C. positive                           var. spinosa                                                                  Neosartorya fischeri M 90 2749 57° C. positive                           var. spinosa                                                                  Neosartorya fischeri M 92 2862 57° C. positive                           var. spinosa                                                                  Neosartorya fischeri M 92 2910 57° C. positive                           var. spinosa                                                                  Neosartorya fischeri M 92 2924 57° C. positive                           var. spinosa                                                                  Neosartorya fischeri M 93 2935 57° C. positive                           var. spinosa                                                                  Neosartorya hiratsukae M 95 3782 57° C. positive                        Neosartorya quadricincta M 93 2984 57° C. positive                      Neosartorya quadricincta M 95 3786 57° C. positive                      Neosartorya spathulata M 93 2978 57° C. positive                        Neosartorya spathulata M 93 2979 57° C. positive                        Neosartorya stramenia M 93 2985 57° C. positive                         Neosartorya tatenoi M 95 3783 57° C. positive                           Paecilomyces carneus M 52 907 57° C. negative                           Paecilomyces carneus M 95 3776 57° C. negative                          Paecilomyces elegans M 79 1670 57° C. negative                          Paecilomyces farinosus M 75 3028 57° C. negative                        Paecilomyces fumoso-roseus M 52 597 57° C. negative                     Paecilomyces inflatus M 57 1387 57° C. negative                         Paecilomyces javanicus M 69 3302 57° C. negative                        Paecilomyces javanicus M 90 2684 57° C. negative                        Paecilomyces lilacinus M 2689 57° C. negative                           Paecilomyces lilacinus M 70 3099 57° C. negative                        Paecilomyces marquandii M 50 85 57° C. negative                         Paecilomyces niphetodes M 79 3238 57° C. negative                       Paecilomyces niphetodes M 95 3778 57° C. negative                       Paecilomyces variotii M 93 3701 57° C. negative                         Paecilomyces zolemiae M 93 3648 57° C. negative                         Penicillium chrysogenum M 47 673 57° C. negative                        Penicillium islandicum M 73 2233 57° C. negative                        Penicillium islandicum M 73 2233 57° C. negative                        Penicillium purpurogenum M 90 2600 57° C. negative                      Penicillium variabile M 78 3162 57° C. negative                         Penicillium viridicatum M 88 2485 57° C. negative                       Talaromyces bacilliformis M 88 1497 57° C. negative                     Talaromyces bacilliformis M 88 2511 57° C. negative                     Talaromyces bacilliformis M 90 1493 57° C. negative                     Talaromyces bacilliformis M 90 2646 57° C. negative                     Talaromyces bacillisporus M 88 2511 57° C. negative                     Talaromyces flavus M 83 1179 57° C. negative                            Talaromyces flavus M 88 2481 57° C. negative                            Talaromyces flavus M 89 1489 57° C. negative                            Talaromyces flavus M 93 2944 57° C. negative                            Talaromyces helicus var. M 88 2510 57° C. negative                       helicus                                                                       Talaromyces luteus M 61 1858 57° C. negative                            Talaromyces sp. M 92 2885 57° C. negative                               Talaromyces sp. M 92 2887 57° C. negative                               Talaromyces sp. M 92 2888 57° C. negative                               Thermoascus crustaceus M 88 2540 57° C. negative                      ______________________________________                                    

The strains obtained from the Paris National Museum of Natural History.

    __________________________________________________________________________     Primer Oligonucleotides specific for Zygosaccharomyces bailii                                             T ° C.                                                                      PCR                                               Genus Species Strain hy. response                                            __________________________________________________________________________     Candida   inconspicua                                                                             M E10   50° C.                                                                      negative                                          Candida magnoliae M E14 50° C. negative                                 Candida mogii M 95 3795 55° C. negative                                 Clavispora lusitaniae M E9 50° C. negative                              Clavispora lusitaniae SIAS 58° C. negative                              Cytofilobasidium  ME13 50° C. negative                                  Debaryomyces hansenii SIAS 58° C. negative                              Debaryomyces hansenii M E18 50° C. negative                             Hyphopichia  M E6 50° C. negative                                       Kluyveromyces marxianus var. M 95 3788 55° C. negative                   lactis                                                                        Kluyveromyces marxianus var. M 95 3789 55° C. negative                   lactis                                                                        Kluyveromyces marxianus var. M 95 3790 55° C. negative                   marxianus                                                                     Kluyveromyces marxianus var. M 95 3791 55° C. negative                   marxianus                                                                     Kluyveromyces var. lactis P 8 57° C. negative                           Moniliella  M E2 50° C. negative                                        Pichia anomala M E12 50° C. negative                                    Pichia anomala SIAS 58° C. negative                                     Pichia fermentans SIAS 58° C. negative                                  Pichia guilliermondii SIAS 58° C. negative                              Pichia guilliermondii M E17 50° C. negative                             Pichia pastoris M 95 3792 55° C. negative                               Pichia pastoris M 95 3793 55° C. negative                               Rhodoturola mucilaginosa M E15 50° C. negative                          Saccharomyces bayanus M 95 3796 55° C. negative                         Saccharomyces bayanus P 562 57° C. negative                             Saccharomyces bayanus P 563 57° C. negative                             Saccharomyces capsularis M 95 3807 55° C. negative                      Saccharomyces cerevisiae H 5035 57° C. negative                         Saccharomyces cerevisiae H 5090 57° C. negative                         Saccharomyces cerevisiae H 5130 57° C. negative                         Saccharomyces cerevisiae H 5160 57° C. negative                         Saccharomyces cerevisiae H 5200 57° C. negative                         Saccharomyces cerevisiae H 5334 57° C. negative                         Saccharomyces cerevisiae M E16 50° C. negative                          Saccharomyces cerevisiae P 1 57° C. negative                            Saccharomyces cerevisiae P 575 57° C. negative                          Saccharomyces cerevisiae SIAS 58° C. negative                           Saccharomyces exiguus M 95 3797 55° C. negative                         Saccharomyces kluyveri M 95 3798 55° C. negative                        Saccharomyces ludwigii M 95 3806 55° C. negative                        Saccharomyces servazii M 95 3799 55° C. negative                        Saccharomyces stellatus M 95 3803 55° C. negative                       Saccharomyces stellatus M 95 3804 55° C. negative                       Saccharomyces unisporus M 95 3805 55° C. negative                       Schyzosaccharo- pombe H 5096 57° C. negative                            myces                                                                          Osmophilic strain  H 11 077 57° C. negative                             Osmophilic strain  H 37 080 57° C. negative                             Torulaspora delbruckii M 95 3794 55° C. negative                        Torulaspora delbruckii P L121 57° C. negative                           Torulaspora delbruckii SIAS 58° C. negative                             Trichosporum capitatum SIAS 50° C. negative                             Zygosaccharomyces bailii M 90 2638 58° C. positive                      Zygosaccharomyces bailii M 90 2639 55° C. positive                      Zygosaccharomyces bailii M 92 2895 55° C. positive                      Zygosaccharomyces bailii M 92 2896 55° C. positive                      Zygosaccharomyces bailii M 92 2897 58° C. positive                      Zygosaccharomyces bailii M 92 2899 58° C. positive                      Zygosaccharomyces bailii M 92 2900 58° C. positive                      Zygosaccharomyces bailii M 92 2901 55° C. positive                      Zygosaccharomyces bailii M 92 2902 55° C. positive                      Zygosaccharomyces bailii M 92 2903 58° C. positive                      Zygosaccharomyces bailii M 92 2904 55° C. positive                      Zygosaccharomyces bailii M 92 2905 55° C. positive                      Zygosaccharomyces bailii M 92 2906 55° C. positive                      Zygosaccharomyces bailii M 92 2907 55° C. positive                      Zygosaccharomyces bailii M 92 2908 58° C. positive                      Zygosaccharomyces bailii M 92 2909 58° C. positive                      Zygosaccharomyces bailii M 92 2911 55° C. positive                      Zygosaccharomyces bailii M 92 2912 58° C. positive                      Zygosaccharomyces bailii M 92 2913 58° C. positive                      Zygosaccharomyces bailii M 92 2914 55° C. positive                      Zygosaccharomyces bailii M 92 2915 58° C. positive                      Zygosaccharomyces bailii M 92 2916 55° C. positive                      Zygosaccharomyces bailii M 92 2917 58° C. positive                      Zygosaccharomyces bailii M 92 2918 58° C. positive                      Zygosaccharomyces bailii M 92 2920 55° C. positive                      Zygosaccharomyces bailii M 92 2921 55° C. positive                      Zygosaccharomyces bailii M 92 2922 58° C. positive                      Zygosaccharomyces bailii M 92 2923 58° C. positive                      Zygosaccharomyces bailii M E11 55° C. positive                          Zygosaccharomyces bailii PL443 58° C. positive                          Zygosaccharomyces bisporus DBVPG 6382 58° C. negative                   Zygosaccharomyces cidri DBVPG 6385 55° C. negative                      Zygosaccharomyces fermentati DBVPG 6297 55° C. negative                 Zygosaccharomyces florentinus DBVPG 6186 55° C. negative                                              Zygosaccharomyces microellipsoides DBVPG                                      6188 55° C. negative                       Zygosaccharomyces mrakii DBVPG 6289 55° C. negative                     Zygosaccharomyces rouxii MJCL 30008 55° C. negative                     Zygosaccharomyces rouxii SIAS 58° C. negative                         __________________________________________________________________________      SOURCE OF THE STRAINS                                                          DEVPG: Department of Plant Biology, Perugia (Italy)                            H: Heudebert Company                                                           M: Paris National Museum of Natural History                                    MUCL: Fungus Culture Collection of the Catholic University of Louvains         P: PernodRicard Company                                                        SIAS: Company SIAS France                                                

Source of the Strains

DBVPG: Department of Plant Biology, Perugia (Italy)

H: Heudebert Company

M: Paris National Museum of Natural History

MUCL: Fungus Culture Collection of the Catholic University of Louvains

P: Pernod-Ricard Company

SIAS: Company SIAS France

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 12                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 186 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - CCGAGTGCGG GTCCCTCGTG CCCCAACCTC CAACCCGTGT TGACCGACAC CT -             #GTTGCTTC     60                                                                  - - GGCGGGCCCG CCCATGGGCT CCCGCCCGGC CGCCGGGGGG CCTCGTCGCC CC -             #CGGGCCCG    120                                                                  - - CCGCCCGCCG AAGACCCCTC GAACGCTGCC TTGAAGGTTG CCGTCTGAGT AT -             #AAAATCAA    180                                                                  - - TCGTTA                 - #                  - #                  -      #          186                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 187 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - GCTAACCCTC CAGCCCGGCT GGTGTGTTGG GCCGCCGTCC CCTCCGGGGG AC -              #GGGCCCGA     60                                                                  - - AAGGCAGCGG CGGCGCCGTC CGGTCCTCGA GCGTATGGGG CTTTGTCACA CG -             #CTCTGGTA    120                                                                  - - GGCCCGGCCG GCTTGCTGGC AAACGACCTC ACGGTCACCT AACTTCTCTC TT -             #AGGTTGAC    180                                                                  - - CTCGGAT                 - #                  - #                        - #         187                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 183 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - CCGAGTGAGG GCCTCTGGGT CCAACTTCCC ACCCGTGTCT ATCGTACCTT GT -             #TGCTTCGG     60                                                                  - - CGGGCCCGCC GTTTCGACGG CCGCCGGGGA GGCCTCGCGC CCCCGGGCCC GC -             #GCCCGCCG    120                                                                  - - AAGACCCCAA CATGAACGCT GTTCTGAAAG TATGCAGTCT GAGTTGATTA TC -             #ATAATCAG    180                                                                  - - TTA                  - #                  - #                  - #                 183                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 176 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - GCTGCCCTCA GGCACGGCTT GTGTGTTGGG CGTCCCCTCT CCCGGGAACG GG -              #CCCGAAAG     60                                                                  - - GCAGCGGCGG CACCGCGTCC GGTCCTCGAG CGTATGGGGC TTTGTCACCC GC -             #TCTGTAGG    120                                                                  - - CCCGGCCGGC GCCAGCCGAC ACCCAACTTT ATTTCTAAGG TTGACCTCGG AT - #CAGG             176                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 279 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - TAGAAAAATT TCTGATTGAC GAGTCTGGAG CAGTTGTGTT CTTTCTGTTT TT -              #TTTAAGGC     60                                                                  - - CTGCGCTTAA TTGCCGTCTA GAGCGGAGGG AGTTAAGCAT AGTTGCCTTT GG -             #CTTTCAAT    120                                                                  - - TTACACACAG TGGAGTTTCT ACTTTTTTTA TTCTTCTTTG GGAGGATGGG TT -             #CGTCCCGC    180                                                                  - - TCCCAGAGGT AAACACAAAC AATTTTTTTT ATTTTATTTT ATTTTATTAT TA -             #TAATAATA    240                                                                  - - ATAATACAGT CAAAACGAAT ACTAAAAAAA AAATATTCA      - #                       - #   279                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 237 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - CCTTCTCAAA CATTCGTGTT TGGTAGTGAG TGATACTCTG TTTTATATTT GG -              #GTTAACTT     60                                                                  - - GAAATTGCAA GCCTTTTGGG ACGCGTGTGG GTGAGTTTTA GGCCGGAAAC GT -             #CTTGCTCT    120                                                                  - - CCTCTTTCCT AACCAAATGT CGTATTAGGT TTTACCGACT CCGACAGACG GG -             #ACTAGGAG    180                                                                  - - ATTGGGTGAG TGATAGCAAT ATCGAGCTCT GCCTAATTTT TTTTTTTGCG CG - #CCTTG            237                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - GGTTGCCGTC TGAGTATAAA ATCAA          - #                  - #                    25                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - GAAGTTAGGT GACCGTGAGG TCGTT          - #                  - #                    25                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - AAGACCCCAA CATGAACGCT GTTCT          - #                  - #                    25                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - TCCGAGGTCA ACCTTAGAAA TAAAGTT          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - TAGAGCGGAG GGAGTTAAGC ATAGT          - #                  - #                    25                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - TGGTTAGGAA AGAGGAGAGC AAGAC          - #                  - #                    25                                                                     __________________________________________________________________________ 

We claim:
 1. A method for detecting the presence or absence of Byssochlamys nivea, Neosartorya fischeri or Zygosaccharomyces bailii in a sample, comprising amplifying the DNA in the sample using a primer composition which specifically hybridizes to the ITS regions of SEQ ID Nos. 1-6, or which specifically hybridizes to the complement of SEQ ID Nos. 1-6, to produce amplified fragments and detecting the presence or absence of the amplified fragments to thereby detect the presence of Byssochlamys nivea, Neosartorya fischeri or Zygosaccharomyces bailii.
 2. The method of claim 1, wherein the primer composition is a primer composition comprising SEQ ID Nos. 7-12.
 3. The process according to claim 1, wherein said amplifying is carried out by the PCR method.
 4. The process according to claim 1, wherein said microorganisms are detected in food products.
 5. The process according to claim 4, wherein said food products are fruit-based products.
 6. The process according to claim 5, wherein a sample containing said fruit-based products is pretreated prior to step (i) so as to liberate and concentrate the spores or the cells, to reduce or suppress the action of the inhibitors of Taq Polymerase, and to extract the DNA from said spores or cells.
 7. The process according to claim 5, wherein said fruit-based products are strawberry-based food products.
 8. The process according to claim 5, wherein samples containing said fruit-based products are treated with a mixture of cellulase/hemicellulase so as to liquefy them and then filtered and centrifuged under conditions which make it possible to obtain a product containing the cells from which the DNA can be extracted.
 9. A primer composition comprising SEQ ID Nos. 7-12.
 10. A kit comprising SEQ ID Nos. 7-12.
 11. A primer composition comprising primers which specifically hybridize to the ITS regions of SEQ ID Nos. 1-6 or to the complementary sequences thereof.
 12. A kit comprising a primer composition according to claim
 7. 